Expression and Regulation of the Arabidopsis thaliana Cel1 Endo 1,4 �� Glucanase Gene During Compatible Plant-Nematode Interactions

The root-knot nematode Meloidogyne incognita is an obligate endoparasite of plant roots and stimulates elaborate modifications of selected root vascular cells to form giant cells for feeding. An Arabidopsis thaliana endoglucanase (Atcel1) promoter is activated in giant cells that were formed in Atcel1::UidA transgenic tobacco and Arabidopsis plants. Activity of the full-length Atcel1 promoter was detected in root and shoot elongation zones and in the lateral root primordia. Different 5’ and internal deletions of regions of the 1,673 bp Atcel1 promoter were each fused to the UidA reporter gene and transformed in tobacco, and roots of the transformants were inoculated with M. incognita to assay for GUS expression in giant cells and noninfected plant tissues. Comparison of the Atcel1 promoter deletion constructs showed that the region between −1,673 and −1,171 (fragment 1) was essential for Atcel1 promoter activity in giant cells and roots. Fragment 1 alone, however, was not sufficient for Atcel1 expression in giant cells or roots, suggesting that cis-acting elements in fragment 1 may function in consort with other elements within the Atcel1 promoter. Root-knot nematodes and giant cells developed normally within roots of Arabidopsis that expressed a functional antisense construct to Atcel1, suggesting that a functional redundancy in endoglucanase activity may represent another level of regulatory control of cell wall-modifying activity within nematode feeding cells.     

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

Background

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.

Methods

Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.

Results

A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.

Conclusion

Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.     

Loosening Xyloglucan Accelerates the Enzymatic Degradation of Cellulose in Wood

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrUs was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.     

A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model.

Nitric oxide (NO), an important cell signaling molecule, is considered a marker of inflammatory response and is elevated in asthmatics. This study investigated the effects of montelukast (a leukotriene receptor antagonist) on iNOS expression and activity in a Brown Norway (BN) rat allergic inflammation model and in L2 lung epithelial cells. Allergic inflammation was induced by ovalbumin (OVA) injection in BN rats followed by treatment with either montelukast or dexamethasone (DX). Allergen inhalation was performed, and post-allergen Penh was measured 5 min after the challenge. Cysteinyl leukotriene levels were measured in bronchoalveolar lavage (BAL) fluid and lung iNOS expression and activity determined. These parameters were also measured in cytokine stimulated L2 lung epithelial cells. iNOS expression was significantly higher in OVA challenged rats compared to the naive, DX, and montelukast treated groups, as confirmed by immunohistochemistry and Western blot analysis. However, no significant differences in NOS activity were found. Cysteinyl leukotriene measured in BAL was significantly higher in all OVA challenged rats compared to naive controls. Incubation of L2 cells with a mixture of interferon gamma (IFNgamma), lipopolysaccharide (LPS), and tumor necrosis factor (TNFalpha) resulted in high levels of nitrite formation resulting from iNOS induction. Treatment of cytokine stimulated cells with DX or montelukast significantly decreased iNOS expression and activity. No detectable cysteinyl leukotrienes were found in the supernatant fluid of L2 cells. This study confirms the ability of montelukast to modulate iNOS function and raises the possibility that changes in iNOS expression and activity may occur via pathways independent of cysteinyl leukotrienes.     

A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity

The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Multiple display of catalytic modules on a protein scaffold: nano-fabrication of enzyme particles

Self assembly is a prerequisite for fabricating nanoscale structures. Here we present a new fusion protein based on the stress-responsive homo-oligomeric protein, SP1. This ring-shaped protein is a highly stable homododecamer, which can be potentially utilized to self-assemble different modules and enzymes in a predicted and oriented manner. For that purpose, a cohesin module (a component of the bacterial cellulosome) was selected, its gene fused in-frame to SP1, and the fusion protein was expressed in Escherichia coli. The cohesin module, specialized to incorporate different enzymes through specific recognition of a dockerin modular counterpart, is used to display new moieties on the SP1 scaffold. The SP1 scaffold displayed 12 active cohesin modules and specific binding to a dockerin-fused cellulase enzyme from Thermobifida fusca. Moreover, we found a significant increase in specific activity of the scaffold-displayed enzymes.     

Characterization of SP1, a stress-responsive,boiling-soluble,homo-oligomeric protein from aspen

In flowering plants, the vegetative nucleus and the two sperm cells are proposed to form a functional assemblage, the male germ unit (MGU). Here, we describe the developmental pathway of MGU assembly in Arabidopsis and report two classes of mutations that affect the integrity and/or the positioning of the MGU in the mature pollen grain. In germ unit malformed (gum) mutants, the vegetative nucleus is positioned adjacent to the pollen grain wall, separate from the two sperm cells, whereas in MGU displaced (mud) mutants, the intact MGU is displaced to the pollen grain wall. mud and gum mutants correspond to male-specific gametophytic mutations that also reduce pollen fitness. Genetic mapping showed that the gum1 and gum2 mutations are genetically linked, possibly allelic, whereas the mud1 and mud2 mutations correspond to two unlinked loci mapping on different chromosomes. The hierarchical relationship between mud and gum mutations was investigated by phenotypic analysis of double mutants. gum1 appeared to act earlier than mud1 and mud2, affecting initial MGU assembly and its stability during pollen maturation. In contrast, mud1 and mud2 mutations appear to act only on MGU positioning during final maturation. From in planta analyses of pollen germination in mud and gum mutants, we conclude that the initial proximity and positioning of MGU components is not required for their entrance into the pollen tube, but the efficiency of MGU translocation is reduced.     

Sugars enhance the expression of gibberellin-induced genes in developing petunia flowers

Sugar is essential for the development of detached Petunia hybrida flowers. We have shown that sucrose (Suc) and gibberellic acid (GA₃) are required for anthocyanin accumulation and the expression of various genes in developing petunia corollas. The effect of GA₃ on the expression of the gibberellin-induced gene and chalcone synthase gene, in detached corollas, was promoted by metabolic sugars such as Suc, glucose (Glc) and fructose, but not by the nonmetabolized 3- O -methylglucose and the sugar alcohol, mannitol. Several pieces of evidence support sugars' signaling role in the corollas and the possible involvement of hexokinase as the sugar sensor. Mannose, which is inefficiently metabolized but is phosphorylated by hexokinase at efficiency similar to Glc, was as effective as Glc in promoting gene expression and pigmentation. 2-Deoxyglucose, which is a substrate for hexokinase but is not metabolized in glycolysis, also promoted gene expression. On the other hand, mannoheptulose, a competitive inhibitor of hexokinase, completely abolished the promotive effect of Glc. We suggest that sugar-phosphorylation-related signal transduction interacts with the gibberellin signal to induce gene expression and anthocyanin accumulation in developing petunia corollas.     

Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).